Nicotinyl alcohol ether derivative, preparation method therefor, and pharmaceutical composition and uses thereof

ABSTRACT

The present invention discloses a nicotinyl alcohol ether derivative, a preparation method therefor, and a pharmaceutical composition and uses thereof. Specifically, the invention relates to nicotinyl alcohol ether derivatives represented by formula (I), a pharmaceutically-acceptable salt thereof, a stereoisomer thereof, a preparation method therefor, a pharmaceutical composition containing the one or more compounds, and uses of the compounds in treating diseases related to PD-1/PD-L1 signal channels, such as cancers, infectious diseases and autoimmune diseases.

FIELD OF THE INVENTION

The present invention discloses a nicotinyl alcohol ether derivative, apreparation method therefor, and a pharmaceutical composition and usesthereof. Specifically, the invention relates to nicotinyl alcohol etherderivatives represented by formula (I), a pharmaceutically-acceptablesalt thereof, a stereoisomer thereof, a preparation method therefor, apharmaceutical composition containing the one or more compounds, anduses of the compounds in treating diseases related to PD-1/PD-L1 signalchannels, such as cancers, infectious diseases and autoimmune diseases.

BACKGROUND OF THE INVENTION

With the deepening of research on cancer immunology, it has been foundthat the tumor microenvironment can protect tumor cells from beingrecognized and killed by the human immune system. The immune escape oftumor cells plays a very important role in tumor occurrence anddevelopment. In 2013, Science magazine ranked tumor immunotherapy as thefirst of the top ten breakthroughs, once again making immunotherapy a“focus” in the field of cancer treatment.

Activation or inhibition of immune cells is regulated by positive andnegative signals, wherein programmed death 1 (PD-1)/PD-1 ligand (PD-L1)is a negative immune regulatory signal that inhibits the immune activityof tumor-specific CD8+ T cells and mediates immune escape.

Tumor cells evade the immune system by the binding of programmed celldeath ligand (PD-L1) produced on its surface to the PD-1 protein of Tcells. The tumor microenvironment induces high expression of PD-1molecules in infiltrating T cells, and tumor cells highly express PD-1ligands PD-L1 and PD-L2, resulting in continuous activation of the PD-1pathway in the tumor microenviroment. The inhibited T cells cannot findthe tumor so that it cannot signal the immune system to attack and killthe tumor cells. The PD-1 antibody against PD-1 or PD-L1 blocks thispathway by preventing the two proteins from binding and partiallyrestores the function of T cells, enabling them to kill tumor cells.

PD-1/PD-L1-based immunotherapy is a new generation high-profileimmunotherapy, aiming to use the body's own immune system to fighttumors. It has the potential to treat multiple types of tumors byblocking the PD-1/PD-L1 signaling pathway to induce apoptosis. Recently,a series of surprising studies have confirmed that PD-1/PD-L1 inhibitoryantibodies have strong anti-tumor activity against a variety of tumors,which is particularly eye-catching. On Sep. 4, 2014, Keytruda®(pembrolizumab) from Merck, USA, became the first FDA-approved PD-1monoclonal antibody for the treatment of advanced or unresectablemelanoma patients who were unresponsive for other medications.Currently, MSD is investigating the potential of Keytruda in more than30 different types of cancer, including various types of blood cancer,lung cancer, breast cancer, bladder cancer, stomach cancer, and head andneck cancer. On Dec. 22, 2014, pharmaceutical giant Bristol-Myers Squibbtook the lead in obtaining accelerated approval from the US Food andDrug Administration (FDA). Its anti-cancer immunotherapy drug nivolumabwas listed under the trade name Opdivo for the treatment of unresectableor metastatic melanoma patients who have not responded to other drugsand it is the second US-listed PD-1 inhibitor after MSD's Keytruda. OnMar. 4, 2015, FDA approved nivolumab for the treatment of metastaticsquamous non-small cell lung cancer that progressed duringplatinum-based chemotherapy or after chemotherapy. According to a PhaseIb KEYNOTE-028 study of the treatment of solid tumors by Keytruda(pembrolizumab) published by MSD, Keytruda treatment achieved a 28%overall response rate (ORR) in 25 patients with pleural mesothelioma(PM). And 48% of patients have stable disease and the disease controlrate has reached 76%. Patients with advanced Hodgkin's lymphoma (HL) whohad no treatment response to any of the approved drugs were able toachieve complete remission after receiving treatment with MSD's Keytrudaand Bristol-Myers' Opdvio. At the 2015 AACR Annual Meeting, Leisha A.Emens, MD, PhD, associate professor of oncology at the Johns HopkinsKimmel Cancer Center, reported that Roche's PD-L1 monoclonal antibodyMPDL3280A has a long-lasting effect in advanced triple-negative breastcancer.

Tumor immunotherapy is considered a revolution in cancer treatment aftertumor targeting therapy. However, the monoclonal antibody therapeuticdrug has its own defects: it is easily decomposed by proteases, so it isunstable within the body and cannot be taken orally; it is easy toproduce immune cross-reaction; the product quality is not easy tocontrol and the production technology is high; a large amount ofpreparation and purification is difficult, and the cost is high; it isinconvenient to use and it only can be injected or drip. Therefore,small molecule inhibitors of PD-1/PD-L1 interaction are a better choicefor tumor immunotherapy.

CONTENTS OF THE INVENTION

The technical problem to be solved by the present invention is toprovide a nicotinyl alcohol ether derivative with the structural formula(I) which inhibits the interaction of PD-1/PD-L1, and a stereoisomerthereof and a pharmaceutically acceptable salt thereof, and apreparation method therefor and medicament compositions thereof andtheir use in the prevention or treatment of a disease associated withthe PD-1/PD-L1 signaling pathway.

The technical solutions below are provided by the present invention inorder to solve the above technical problem.

The first aspect of the technical solution is to provide a nicotinylalcohol ether derivative represented by formula (I), a stereoisomerthereof and a pharmaceutically-acceptable salt thereof:

wherein:R₁ is selected from

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sufydryl,imidazolyl thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, methoxy.

Preferable are nicotinyl alcohol ether derivatives, stereoisomersthereof and pharmaceutically acceptable salts thereof, wherein thecompound is represented by formula (IA):

wherein:R₁ is selected from

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are nicotinyl alcohol ether derivatives, stereoisomersthereof and pharmaceutically acceptable salts thereof, wherein thecompound is represented by formula (IA-1):

wherein:R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are nicotinyl alcohol ether derivatives, stereoisomersthereof and pharmaceutically acceptable salts thereof, wherein thecompound is represented by formula (IA-2):

wherein:R₃ is selected from substituted C₁-C₈ saturated alkylamino, substitutedC₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substitutedwith substituent(s) selected from hydrogen, fluorine, chlorine, bromine,iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino,acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂),ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂),sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino(—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl,imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄alkyl, ethenyl, trifluoromethyl, and methoxy.

Preferable are nicotinyl alcohol ether derivatives, stereoisomersthereof and pharmaceutically acceptable salts thereof, wherein thecompound is represented in the above formulae, wherein R₃ is selectedfrom:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl,pentyl, hexyl, heptyl, octyl; andX is selected from hydrogen, fluorine, chlorine, bromine, methyl,ethenyl, and trifluoromethyl.

Most preferable compounds are selected from the following: Ethyl

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate dihydrochloride

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serine

-   (S)-Ethyl    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate

-   (N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzyl) serine

-   -   (S)-Ethyl        N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)        serinate dihydrochloride

-   (S)-isopropyl    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate dihydrochloride

-   (R)-Ethyl    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate

-   (R)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzyl) serine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    glycine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    valine

-   (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamino) but-2-enenitrile

-   N,    N-bis(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamine

-   N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamine

-   N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamine

-   (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamino) but-2-enoic acid

-   2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamino) ethanesulfonic acid

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    leucine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    tyrosine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    isoleucine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    asparagine

-   N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)    benzylamine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    alanine

-   N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    proline

-   (S)-Sodium    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate

-   (S)-Calcium    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate

-   (S)-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl    N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)    serinate

The nicotinyl alcohol ether derivative of the above formulae, astereoisomer thereof and a pharmaceutically acceptable salt thereof, arecharacterized in that, the pharmaceutically acceptable salt comprises asalt formed with an inorganic acid, a salt formed with an organic acidsalt, alkali metal ion salt, alkaline earth metal ion salt or a saltformed with organic base which provides a physiologically acceptablecation, and an ammonium salt.

Said inorganic acid is selected from hydrochloric acid, hydrobromicacid, phosphoric acid or sulfuric acid; said organic acid is selectedfrom methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid,citric acid, maleic acid, tartaric acid, fumaric acid, citric acid orlactic acid; said alkali metal ion is selected from lithium ion, sodiumion, potassium ion; said alkaline earth metal ion is selected fromcalcium ion, magnesium ion; said organic base, which providesphysiologically acceptable cation, is selected from methylamine,dimethylamine, trimethylamine, piperidine, morpholine ortris(2-hydroxyethyl)amine.

The second aspect of the present invention provides a method forpreparing the compounds of the first aspect.

For the preparation of the compounds of the formula (I), according toits structure, the preparation method is divided into five steps.

-   -   (a) 2-bromo-3-iodotoluene 1 and benzene boronic acid or        substituted benzene boronic acid or boric acid ester of benzene        or substituted benzene as starting materials are reacted via        suzuki coupling reaction to obtain Intermediate compound 2;    -   (b) intermediate 2 as a starting material is subjected to        bromination of the methyl group by a bromination reagent to give        the bromo intermediate 3;    -   (c) intermediate 3 as a starting material is reacted with        2,4-dihydroxy-X-substituted benzaldehyde under basic conditions        to obtain benzyl aryl ether intermediate 4;    -   (d) intermediate 4 as a starting material is reacted with        pyridin-3-yl-methylene halide under basic conditions to give        intermediate compound 5;    -   (e) an aldehyde group-containing intermediate compound 5 as a        starting material is condensed with an amino group- or an imino        group-containing HR₃ and the resultant product is reduced to        obtain the target compound I.        R₁, R₃ and X each is defined as described in the first aspect.

In addition, the starting materials and intermediates in the abovereaction are obtained easily, and the each step reaction can beperformed easily according to the reported literature or by a skilledworker in the art by a conventional method in organic synthesis. Thecompound of formula I may exist in solvated or unsolvated forms, andcrystallization from different solvents may result in differentsolvates. The pharmaceutically acceptable salts of the formula (I)include different acid addition salts, such as the acid addition saltsof the following inorganic or organic acids: hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid,p-toluenesulfonic acid, Trifluoroacetic acid, citric acid, maleic acid,tartaric acid, fumaric acid, citric acid, lactic acid. Thepharmaceutically acceptable salts of formula I also include variousalkali metal salts such as lithium, sodium, potassium salts; variousalkaline-earth metal salts such as calcium, magnesium salts and ammoniumsalts; and various organic base salts which provide physiologicallyacceptable cations, such as methylamine, dimethylamine, trimethylamine,piperidine, morpholine salts and tris(2-hydroxyethyl)amine salts. All ofthese salts within the scope of the invention can be prepared byconventional methods. During the preparation of the compounds of theformula (I) and their solvates or salts, polycrystalline or eutectic mayoccur under different crystallization conditions.

The third aspect of the present invention provides a pharmaceuticalcomposition comprising which includes the nicotinyl alcohol etherderivative of the first aspect of the present invention and astereoisomer thereof, and the pharmaceutically acceptable salt as anactive ingredient and a pharmaceutically acceptable carrier orexcipient.

The invention further relates to a pharmaceutical composition comprisinga compound of the invention as an active ingredient. The pharmaceuticalcomposition can be prepared according to methods well known in the art.Any dosage form suitable for human or animal use can be prepared bycombining a compound of the invention with one or more pharmaceuticallyacceptable excipients and/or adjuvants in solid or liquid. The contentof the compound of the present invention in its pharmaceuticalcomposition is usually from 0.1 to 95% by weight.

The compound of the present invention or the pharmaceutical compositioncontaining the same can be administered in a unit dosage form, viaenteral or parenteral route, such as oral, intravenous, intramuscular,subcutaneous, nasal, oral mucosa, eye, lung and the respiratory tract,skin, vagina, rectum, etc.

The dosage form can be a liquid dosage form, a solid dosage form or asemi-solid dosage form. Liquid dosage forms can be solution (includingtrue solution and colloidal solution), emulsion (including o/w type, w/otype and double emulsion), suspension, injection (including waterinjection, powder injection and infusion), eye drops, nasal drops,lotions, liniments, etc.; solid dosage forms may be tablets (includingordinary tablets, enteric tablets, lozenges, dispersible tablets,chewable tablets, effervescent tablets, orally disintegrating tablets),capsules (including hard capsules, soft capsules, enteric capsules),granules, powders, pellets, dropping pills, suppositories, films,patches, gas (powder) sprays, sprays, etc.; semi-solid dosage forms canbe ointments, gel, paste, etc.

The compounds of the present invention can be formulated into commonpreparations, as well as sustained release preparations, controlledrelease preparations, targeted preparations, and various microparticledelivery systems.

In order to form tablets of the compound of the present invention into,various excipients known in the art, including diluents, binders,wetting agents, disintegrating agents, lubricants, and glidants, can beused widely. The diluent may be starch, dextrin, sucrose, glucose,lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose,calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.;the wetting agent may be water, ethanol, or isopropanol, etc.; thebinder may be starch syrup, dextrin, syrup, honey, glucose solution,microcrystalline cellulose, acacia mucilage, gelatine, sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, ethyl cellulose, acrylic resin, carbomer,polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can bedry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked poly vinyl pyrrolidone, croscarmellose sodium,sodium carboxymethyl starch, sodium hydrogencarbonate and citric acid,polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate,etc.; lubricant and glidant may be talc, silica, stearate, tartaricacid, liquid paraffin, polyethylene glycol, etc.

Tablets may also be further formulated into coated tablets such as sugarcoated tablets, film-coated tablets, enteric coated tablets, or bilayertablets and multilayer tablets.

In order to prepare the dose unit as a capsule, the active ingredientcompound of the present invention may be mixed with a diluent, aglidant, and the mixture may be directly placed in a hard capsule or asoft capsule. The active ingredient can also be formulated into agranule or pellet with a diluent, a binder, a disintegrant, and thenplaced in a hard or soft capsule. Various diluents, binders, wettingagents, disintegrating agents and glidants for preparing the tablets ofthe compound of the invention can also be used to prepare the capsulesof the compound of the invention.

In order to prepare the compound of the present invention as aninjection, water, ethanol, isopropanol, propylene glycol or theirmixture may be used as a solvent. In addition, an appropriate amount ofa solubilizing agent, a co-solvent, a pH adjusting agent, and an osmoticpressure adjusting agent which are commonly used in the art can beadded. The solubilizing agent or co-solvent may be poloxamer, lecithin,hydroxypropyl-β-cyclodextrin, etc.; the pH adjusting agent may bephosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; osmoticpressure regulating agent may be sodium chloride, mannitol, glucose,phosphate, acetate, etc. For preparing a lyophilized powder injection,mannitol, glucose and so on may also be added as a proppant.

In addition, coloring agents, preservatives, perfumes, flavoring agentsor other additives may also be added to the pharmaceutical preparationsas needed. The compound or pharmaceutical composition of the presentinvention can be administered by any known administration method for thepurpose of administration and enhancing the therapeutic effect.

The dosage of the compound or the pharmaceutical composition of thepresent invention can be administered in a wide range depending on thenature and severity of the disease to be prevented or treated, theindividual condition of the patient or animal, the route ofadministration and the dosage form, etc. In general, a suitable dailydose of the compound of the invention will range from 0.001 to 150 mg/kgbody weight, preferably from 0.01 to 100 mg/kg body weight.

The above dosages may be administered in a single dosage unit or individed dose units depending on the clinical experience of the physicianand the dosage regimen including the use of other therapeutic means.

The compounds or compositions of the invention may be administered aloneor in combination with other therapeutic or symptomatic agents. When thecompound of the present invention synergizes with other therapeuticagents, its dosage should be adjusted according to the actual situation.

The fourth aspect of the present invention provides a nicotinyl alcoholether derivative, or a stereoisomer thereof, or a pharmaceuticallyacceptable salt thereof, which are used for the preparation of amedicament useful for preventing and/or treating a disease associatedwith the PD-1/PD-L1 signaling pathway.

The disease associated with the PD-1/PD-L1 signaling pathway is selectedfrom cancer, infectious diseases, and autoimmune diseases. The cancer isselected from skin cancer, lung cancer, urinary tumor, hematologicaltumor, breast cancer, glioma, digestive system tumor, reproductivesystem tumor, lymphoma, nervous system tumor, brain tumor, head and neckcancer. The infectious disease is selected from bacterial infection andviral infection. The autoimmune disease is selected from organ-specificautoimmune disease, systemic autoimmune disease, wherein theorgan-specific autoimmune disease includes chronic lymphocyticthyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus,myasthenia gravis, ulcerative colitis, malignant anemia with chronicatrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primarybiliary cirrhosis, multiple cerebrospinal sclerosis, and acuteidiopathic polyneuritis. And the systemic autoimmune diseases includerheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis,scleroderma, pemphigus, dermatomyositis, mixed connective tissuedisease, autoimmune hemolytic anemia.

BENEFICIAL TECHNICAL EFFECTS

The compounds of the present invention have high inhibitory activity onPD-1/PD-L1 interaction, much higher than the reported compounds. Theyhave strong ability of binding PD-L1 protein, and the kD value ofaffinity can reach 2.025E-11, even stronger than the reported antibodiesof PD-L1. These compounds also have the ability to relieve theinhibition of IFN-γ by PD-L1, whose IC₅₀ value can reach 1.8×10⁻¹⁰ mol/Llevel. The pharmacodynamic studies in vivo show that the compounds cansignificantly inhibit the growth of subcutaneous tumors in both tumorvolume and weight. The number of lymphocytes in blood and spleen of micecan be increased obviously.

EXAMPLES

The invention is further illustrated by the following examples; however,the invention is not limited by the illustrative examples set hereinbelow.

Measuring instrument: Nuclear magnetic resonance spectroscopy wascarried out by using a Vaariaan Mercury 300 nuclear magnetic resonanceapparatus. Mass spectrometry was performed by using ZAD-2F massspectrometer and VG300 mass spectrometer.

Example 1 EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride

2-Bromo-3-methyl-1, 1′-biphenyl

To a 100 ml flask were added 2-bromo-3-iodotoluene (700 mg) anddioxane/water (volume ratio 5/1) with stirring. The solution was bubbledwith argon for 10 min to remove dissolved oxygen. Then, phenylboronicacid (350 mg), cesium carbonate (1800 mg), and triphenylphosphinepalladium (80 mg) were sequentially added. The mixture was stirred for12 h at 80-100° C. under argon protection. The reaction was stopped.After cooling to room temperature, the mixture was filtered withdiatomaceous earth. The filtrate was concentrated under reduced pressureand extracted with water and ethyl acetate for three times. The organicphase was combined, washed with saturated brine, and dried overanhydrous sodium sulfate. The organic layer was filtered and evaporatedunder reduced pressure to dryness to afford 2-bromo-3-methyl-1,1′-biphenyl as colorless oil (480 mg). ¹H NMR (400 MHz, DMSO-d₆), δ7.49-7.29 (m, 7H, Ar—H), 7.14 (d, 1H, Ar—H), 2.42 (s, 3H, Ar—CH₃). MS(FAB): 248 (M+1).

2-Bromo-3-(bromomethyl)-1,1′-biphenyl

2-Bromo-3-methyl-1,1′-biphenyl (450 mg) as a starting material wasweighed and was dissolved in 40 ml of CCl₄ in a 100 ml flask. To thissolution was added NBS (360 mg) while stirring. And the mixture waswarmed to 80° C. and refluxed. Then benzoyl peroxide (8 mg) was added,and after 2 h, benzoyl peroxide (8 mg) was added again, and the reactionwas continued for another 2 h. The reaction was stopped. After coolingto room temperature, the mixture was quenched with water, extracted withdichloromethane and water. The organic phase was washed with saturatedbrine, and dried over anhydrous sodium sulfate. The organic layer wasfiltered and evaporated under reduced pressure to dryness to afford2-bromo-3-(bromomethyl)-1, 1′-biphenyl as yellow oil (380 mg), which wasused for the next step without further purification.

4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde

5-chloro-2, 4-dihydroxybenzaldehyde (160 mg) was weighed and dissolvedin 12 ml of anhydrous acetonitrile in a 50 ml flask, and sodium hydrogencarbonate (200 mg) was added. After stirring at room temperature for 40min, 2-bromo-3-phenylbenzyl bromide (380 mg, dissolved in 16 ml of DMF)was slowly added dropwise to the reaction mixture via a constantpressure dropping funnel, and heated to reflux until the reaction wascompleted. After cooling to room temperature, the mixture was extractedwith water and ethyl acetate. The organic phase was washed withsaturated brine, and dried over anhydrous sodium sulfate, then filtratedand evaporated under reduced pressure to dryness. The crude residue waspurified by silica gel column chromatography to afford4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxy benzaldehyde (300 mg)as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.99 (s, 1H, —OH), 10.03(s, 1H, —CHO), 7.64 (d, 1H, Ar—H), 7.57 (d, 1H, Ar—H), 7.45 (m, 4H,Ar—H), 7.37 (d, 2H, Ar—H), 6.67 (d, 1H, Ar—H), 6.59 (s, 1H, Ar—H), 5.25(s, 2H, —CH₂—). MS (FAB): 418 (M+1).

4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde

4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde (100 mg)was dissolved in 6 ml of DMF in a 50 ml flask, and then cesium carbonate(127.53 mg) was added. After stirring at room temperature for 15 min, asolution of 3-bromomethylenepyridine (76.65 mg) in DMF (4 ml) was addeddropwise. After the mixture was stirred at 80° C. for 2 h, the reactionwas stopped. After cooling to room temperature, the mixture wasextracted with water and ethyl acetate. The organic phase was washedwith saturated brine, and dried over anhydrous sodium sulfate, thenfiltrated and evaporated under reduced pressure to dryness. The cruderesidue was purified by silica gel column chromatography to afford awhite solid (70 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H, —CHO),8.00 (s, 1H, Ar—H), 7.83 (dd, 2H, Ar—H), 7.72 (d, 1H, Ar—H), 7.61 (t,2H, Ar—H), 7.55-7.23 (m, 6H, Ar—H), 6.95 (s, 1H, Ar—H), 6.81 (d, 1H,Ar—H), 5.35 (s, 2H, —CH₂—), 5.30 (s, 2H, —CH₂—). MS (FAB): 510 (M+1).

EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride

4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde(80 mg) was dissolved in 5 ml of DMF, and then racemic ethyl ester ofserine (59 mg) and acetic acid glacial (57 mg) were added. Afterstirring at room temperature for 20 min, sodium cyanoborohydride (25 mg)was added and the mixture was stirred at 25° C. for 14 h. The reactionwas stopped. The mixture was extracted with water and ethyl acetate. Theorganic phase was washed with saturated brine, and dried over anhydroussodium sulfate, then filtrated and evaporated under reduced pressure todryness. The residue was dissolved in ethanol, heated to reflux untilthe reaction was complete. The mixture was evaporated to dryness. Thecrude residue was purified by silica gel column chromatography to affordethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate (60 mg) as yellow oil. The product was then reacted with asolution of hydrogen chloride in ethanol to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ9.65 (s, 1H, —HCl), 9.41 (s, 1H, —HCl), 9.06 (s, 1H, —ArH), 8.88-8.76(m, 1H, —ArH), 8.56 (d, J=7.9 Hz, 1H, —ArH), 8.01-7.86 (m, 1H, —ArH),7.64 (d, J=13.4 Hz, 2H, —ArH), 7.56-7.30 (m, 7H, —ArH), 7.12 (s, 1H,—ArH), 5.42 (s, 2H, —CH₂—), 5.32 (s, 2H, —CH₂—), 4.18 (s, 2H, —CH₂—),4.13-4.07 (m, 1H, —CH—), 4.04 (m, 2H, —CH₂—), 3.94 (dd, 1H, —CH₂—), 3.82(dd, 1H, —CH₂—), 1.15 (t, J=7.1 Hz, 3H, —CH₃). MS (FAB): 626 (M).

Example 2N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine

EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate as pale yellow oil (60 mg) was dissolved in methanol/H₂O (4ml/1 ml), and then lithium hydroxide monohydrate (20 mg) was added.After stirring at room temperature for 2 h, a few drops of acetic acidwere added to the mixture in an ice bath to adjust the pH to acidity.The mixture was filtrated under reduced pressure to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine (45 mg) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (s,1H, —ArH), 8.55 (d, 1H, —ArH), 7.97 (d, 1H, —ArH), 7.65 (d, 1H, —ArH),7.54-7.51 (m, 2H, —ArH), 7.47 (d, 2H, —ArH), 7.44 (dl H, —ArH),7.42-7.40 (m, 2H, —ArH), 7.39-7.36 (m, 2H, —ArH), 7.11 (s, 1H, —ArH),5.32 (s, 2H, —CH₂—), 5.28 (m, 2H, —CH₂—), 3.94 (s, 2H, —CH₂—), 3.69 (dd,1H, —CH₂—), 3.62 (dd, 1H, —CH₂—), 3.16 (t, 1H, —CH—). MS (FAB): 599(M+1).

Example 3 (S)-EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate

The procedure was the same as in Example 1, except that ethyl ester ofL-serine was used in place of racemic ethyl ester of serine to afford(S)-EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J=2.1Hz, 1H, —ArH), 8.55 (dd, J=4.8, 1.7 Hz, 1H, —ArH), 7.88 (d, J=7.9 Hz,1H, —ArH), 7.65 (dd, J=7.6, 1.7 Hz, 1H, —ArH), 7.54-7.45 (m, 3H, —ArH),7.45-7.35 (m, 6H, —ArH), 7.06 (s, 1H, —ArH), 5.30 (s, 2H, —CH₂—), 5.24(s, 2H, —CH₂—), 4.81 (t, J=5.8 Hz, 1H, —CH—), 3.99 (q, J=7.1 Hz, 2H,—CH₂—), 3.74-3.57 (m, 2H, —CH₂—), 3.54 (t, J=5.6 Hz, 2H, —CH₂—), 1.12(t, J=7.1 Hz, 3H, —CH₃). MS (FAB): 626 (M).

Example 4(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine

The procedure was the same as in Example 2, except that (S)-EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinatewas used in place of racemic EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate to afford(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (s, 1H, —ArH),8.55 (d, J=4.7 Hz, 1H, —ArH), 7.97 (d, J=7.9 Hz, 1H, —ArH), 7.65 (d,J=8.7 Hz, 1H, —ArH), 7.54-7.51 (m, 2H, —ArH), 7.47 (d, J=7.7 Hz, 2H,—ArH), 7.44 (d, J=1.7 Hz, 1H, —ArH), 7.42-7.40 (m, 2H, —ArH), 7.39-7.36(m, 2H, —ArH), 7.11 (s, 1H, —ArH), 5.32 (s, 2H, —CH₂—), 5.28 (m, 2H,—CH₂—), 3.94 (s, 2H, —CH₂—), 3.69 (dd, J=11.1, 4.7 Hz, 1H, —CH₂—), 3.62(dd, J=11.1, 6.2 Hz, 1H, —CH₂—), 3.16 (t, J=5.4 Hz, 1H, —CH—). MS (FAB):599 (M+1). [α]²⁷ _(D)=−1.84 (C=0.434, DMSO).

Example 5 (S)-EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride

(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine (598 mg) and 60 ml of anhydrous ethanol were placed in a100 ml round-bottom flask. To the mixture were added 6 ml ofdichlorosulfoxide and two drops of DMF with stirring in an ice waterbath. And after the mixture was stirred at room temperature for 2 h itwas heated to reflux until the reaction was completed. The mixture wasconcentrated under reduced pressure to remove the solvent and afford(S)-ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ9.65 (s, 1H, —HCl), 9.41 (s, 1H, —HCl), 9.06 (s, 1H, —ArH), 8.88-8.76(m, 1H, —ArH), 8.56 (d, J=7.9 Hz, 1H, —ArH), 8.01-7.86 (m, 1H, —ArH),7.64 (d, J=13.4 Hz, 2H, —ArH), 7.56-7.30 (m, 7H, —ArH), 7.12 (s, 1H,—ArH), 5.42 (s, 2H, —CH₂—), 5.32 (s, 2H, —CH₂—), 4.18 (s, 2H, —CH₂—),4.13-4.07 (m, 1H, —CH—), 4.04 (m, 2H, —CH₂—), 3.94 (dd, J=12.1, 2.9 Hz,1H, —CH₂—), 3.82 (dd, J=12.1, 3.8 Hz, 1H, —CH₂—), 1.15 (t, J=7.1 Hz, 3H,—CH₃). MS (FAB): 626 (M). [α]²⁷ _(D)=−1.90 (C=0.422, ethanol).

Example 6 (S)-isopropylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride

(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine (598 mg) and 60 ml of anhydrous isopropanol were placedin a 100 ml round-bottom flask. To the mixture were added 6 ml ofdichlorosulfoxide and two drops of DMF with stirring in an ice waterbath. And after the mixture was stirred at room temperature for 2 h itwas heated to reflux until the reaction was completed. The mixture wasconcentrated under reduced pressure to remove the solvent and afford(S)-isopropylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate dihydrochloride as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.62 (s, 1H, —HCl), 9.40 (s, 1H, —HCl), 9.10 (s, 1H, —ArH), 8.86 (d, 1H,—ArH), 8.59 (d, J=7.6 Hz, 1H, —ArH), 8.01-7.91 (m, 1H, —ArH), 7.73-7.64(m, 2H, —ArH), 7.57-7.46 (m, 3H, —ArH), 7.46-7.37 (m, 4H, —ArH), 7.16(s, 1H, —ArH), 5.47 (s, 2H, —CH₂—), 5.36 (s, 2H, —CH₂—), 4.92 (ml H,—CH—), 4.30-4.15 (m, 2H, —CH₂—), 4.05 (s, 1H, —CH—), 3.97 (dd, J=12.0,3.0 Hz, 1H, —CH₂—), 3.84 (dd, J=12.0, 3.8 Hz, 1H, —CH₂—), 1.20 (d, J=6.4Hz, 3H, —CH₃), 1.18 (d, J=6.4 Hz, 3H, —CH₃). MS (FAB): 640 (M). [α]²⁷_(D)=−5.33 (C=0.075, methanol).

Example 7 (R)-EthylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate

The procedure was the same as in Example 1, except that ethyl ester ofD-serine was used in place of racemic ethyl ester of serine to afford(R)-ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, 1H,—ArH), 8.55 (dd, 1H, —ArH), 7.88 (d, 1H, —ArH), 7.65 (dd, J=7.6, 1.7 Hz,1H, —ArH), 7.54-7.45 (m, 3H, —ArH), 7.45-7.35 (m, 6H, —ArH), 7.06 (s,1H, —ArH), 5.30 (s, 2H, —CH₂—), 5.24 (s, 2H, —CH₂—), 4.81 (t, J=5.8 Hz,1H, —CH—), 3.99 (q, J=7.1 Hz, 2H, —CH₂—), 3.74-3.57 (m, 2H, —CH₂—), 3.54(t, 2H, —CH₂—), 1.12 (t, J=7.1 Hz, 3H, —CH₃). MS (FAB): 626 (M).

Example 8(R)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

The procedure was the same as in Example 2, except that (R)-ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate was used in place of racemic ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate to afford(R)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (s, 1H,—ArH), 8.55 (d, J=4.7 Hz, 1H, —ArH), 7.97 (d, J=7.9 Hz, 1H, —ArH), 7.65(d, J=8.7 Hz, 1H, —ArH), 7.54-7.51 (m, 2H, —ArH), 7.47 (d, J=7.7 Hz, 2H,—ArH), 7.44 (d, J=1.7 Hz, 1H, —ArH), 7.42-7.40 (m, 2H, —ArH), 7.39-7.36(m, 2H, —ArH), 7.11 (s, 1H, —ArH), 5.32 (s, 2H, —CH₂—), 5.28 (m, 2H,—CH₂—), 3.94 (s, 2H, —CH₂—), 3.69 (dd, J=11.1, 4.7 Hz, 1H, —CH₂—), 3.62(dd, J=11.1, 6.2 Hz, 1H, —CH₂—), 3.16 (t, J=5.4 Hz, 1H, —CH—). MS (FAB):599 (M+1). [α]

Example 9(S)—N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

The procedure was the same as in Example 1, except that4-(2-chloro-3-phenylbenzyloxy)-5-chloro-(pyridin-3-yl-methyleneoxy)benzaldehyde was used in place of4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde, and ethyl ester of L-serine was used in place of racemicethyl ester of serine to afford (S)-ethylN-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate.

The procedure was the same as in Example 2, except that (S)-ethylN-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate was used in place of ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate to afford(S)—N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.69 (s,1H, —ArH), 8.51 (d, J=4.6 Hz, 1H, —ArH), 7.93 (d, J=7.6 Hz, 1H, —ArH),7.63 (d, J=7.4 Hz, 1H, —ArH), 7.54-7.33 (m, 9H, —ArH), 7.12 (s, 1H,—ArH), 5.31 (s, 2H, —CH₂—), 5.25 (m, 2H, —CH₂—), 3.91 (s, 2H, —CH₂—),3.66 (dd, J=11.0, 4.0 Hz, 1H, —CH₂—), 3.59 (dd, J=11.0, 6.0 Hz, 1H,—CH₂—), 3.14 (t, J=4.8 Hz, 1H, —CH—). MS (FAB): 554 (M+1).

Example 10(S)—N-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

The procedure was the same as in Example 1, except that4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde was used in place of4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde, and ethyl ester of L-serine was used in place of racemicethyl ester of serine to afford (S)-ethylN-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate.

The procedure was the same as in Example 2, except that (S)-ethylN-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate wasused in place of ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate to afford(S)—N-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.72 (dd,J=8.0, 1.9 Hz, 1H, —ArH), 8.56 (dd, J=4.8, 1.5 Hz, 1H, —ArH), 7.97 (d,J=7.9 Hz, 1H, —ArH), 7.72-7.30 (m, 10H, —ArH), 7.16 (d, J=15.1 Hz, 1H,—ArH), 5.35 (d, J=6.7 Hz, 2H, —CH₂—), 5.27 (dd, J=15.2, 3.2 Hz, 2H,—CH₂—), 3.95 (s, 2H, —CH₂—), 3.70 (dd, J=11.2, 4.5 Hz, 1H, —CH₂—), 3.63(dd, J=11.2, 6.2 Hz, 1H, —CH₂—), 3.18 (t, J=5.3 Hz, 1H, —CH—). MS (FAB):538 (M+1).

Example 11(S)—N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine

The procedure was the same as in Example 1, except that4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehydewas used in place of 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde, and ethyl esterof L-serine was used in place of racemic ethyl ester of serine to afford(S)-ethylN-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate.

The procedure was the same as in Example 2, except that (S)-ethylN-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate was used in place of ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate to afford(S)—N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serine as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, Ar—H),8.50 (s, 1H, Ar—H), 7.91 (d, J=5.6 Hz, 1H, Ar—H), 7.75 (s, 1H, Ar—H),7.61 (d, J=13.2 Hz, 3H, Ar—H), 7.44 (d, J=15.2 Hz, 5H, Ar—H), 7.36 (d,J=8.4 Hz, 2H, Ar—H), 7.09 (s, 1H, Ar—H), 5.29 (s, 2H, —CH₂—), 5.19 (m,2H, —CH₂—), 3.90 (s, 2H, —CH₂—), 3.69-3.55 (m, 2H, —CH₂—), 3.12 (m, 1H,—CH—). MS (FAB): 520 (M+1).

Example 12N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)glycine

(1) 2-bromo-3-phenyltoluene

2-Bromo-3-iodotoluene (350 mg) was placed in 50 ml flask anddioxane/water was added with stirring. And the solution was bubbled withargon for 10 min to remove the dissolved oxygen. Then, phenylboronicacid (172.65 mg), cesium carbonate (961.2 mg) and triphenylphosphinepalladium (40.91 mg) were added and the resulting mixture was stirred at80-100° C. for 12 h under protection of argon. The reaction was stopped.After cooling to room temperature, the mixture was filtered withdiatomaceous earth. The filtrate was concentrated under reduced pressureand extracted with water and ethyl acetate for three times. The organicphase was combined, washed with saturated brine and dried by anhydrousNa₂SO₄. The solution was filtered and evaporated under reduced pressureto dryness. The residue was purified by silica gel column chromatography(petroleum ether) to give colorless oil (221 mg). ¹H NMR (400 MHz,DMSO-d₆), δ 7.49-7.29 (m, 7H, Ar—H), 7.14 (d, 1H, Ar—H), 2.42 (s, 3H,Ar—CH₃).

(2) 2-bromo-3-(bromomethyl)-1,1′-biphenyl

2-Bromo-3-phenyltoluene (234 mg) was weighed and placed in a 100 mlflask and 20 ml of CCl₄ was added. After 2-Bromo-3-phenyltoluene wascompletely dissolved, NBS (178 mg) was added under stirring. And theresulting mixture was heated to 80° C. to reflux. Then benzoyl peroxide(4 mg) was added and another 4 mg of benzoyl peroxide was added after 2h, and the reaction was continued for another 2 h. The reaction wasstopped. The reaction was cooled to room temperature and a suitableamount of water was added and the mixture was extracted withdichloromethane. The organic phase was washed with saturated brine anddried by anhydrous Na₂SO₄. The solution was filtered and evaporatedunder reduced pressure to dryness to give yellow oil (192 mg). Thematerial was used for the next step without further purification.

(3) 2-hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde

To anhydrous acetonitrile (6 ml) in 50 ml flask was added2,4-dihydroxy-5-chlorobenzaldehyde (73.94 mg) and sodium bicarbonate(98.88 mg). After stirring 40 min at room temperature, the solution of2-bromo-3-(bromomethyl)-1,1′-biphenyl (192 mg) in 8 ml of DMF was slowlyadded dropwise to the reaction mixture via a constant pressure droppingfunnel. The resulting mixture was refluxed until the reaction completed.After cooling to room temperature, the mixture was extracted with waterand ethyl acetate. The organic phase was washed with saturated brine,dried over anhydrous sodium sulfate, filtered and evaporated underreduced pressure to dryness. The crude residue was purified by silicagel column chromatography to afford2-hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde (152 mg) as awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.18 (s, 1H, —OH), 10.09 (s,1H, —CHO), 7.74 (s, 1H, —ArH), 7.66 (d, 1H, —ArH), 7.57 (t, 1H, —ArH),7.51 (m, 2H, —ArH), 7.46 (d, 1H, —ArH), 7.42 (d, 3H, —ArH), 6.85 (s, 1H,—ArH), 5.37 (s, 2H, —CH₂—).

(4) 4-(2-bromo-3-phenylbenzoxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde

2-Hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde (100 mg) wasdissolved in 6 ml of DMF in a 50 ml flask, and then cesium carbonate(127.53 mg) was added. After stirring at room temperature for 15 min, asolution of 3-bromomethyleneoxy pyridine (76.65 mg) in DMF (4 ml) wasadded dropwise. After the mixture was stirred at 80° C. for 2 h, thereaction was stopped. After cooling to room temperature, the mixture wasextracted with water and ethyl acetate. The organic phase was washedwith saturated brine, and dried over anhydrous sodium sulfate, thenfiltrated and evaporated under reduced pressure to dryness. The cruderesidue was purified by silica gel column chromatography to afford awhite solid (60 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 10.14 (s, 1H, —CHO),8.71 (d, J=1.6 Hz, 1H, —ArH), 8.54 (d, J=4.8 Hz, 1H, —ArH), 7.94 (d,J=7.8 Hz, 1H, —ArH), 7.83 (s, 1H, —ArH), 7.66 (d, J=7.6 Hz, 1H, —ArH),7.50 (t, J=7.6 Hz, 1H, —ArH), 7.48-7.32 (m, 7H, —ArH), 7.20 (s, 1H,—ArH), 5.42 (s, 2H, —CH₂—), 5.41 (s, 2H, —CH₂—).

(5)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycine

4-(2-bromo-3-phenylbenzoxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde (80 mg) was dissolved in 5 ml of DMF, and then ethylglycinate (49 mg, 0.472 mmol) and acetic acid glacial (57 mg) wereadded. After stirring at room temperature for 20 min, sodiumcyanoborohydride (25 mg) was added and the mixture was stirred at 25° C.for 14 h. The reaction was stopped. The mixture was extracted with waterand ethyl acetate. The organic phase was washed with saturated brine,and dried over anhydrous sodium sulfate, then filtrated and evaporatedunder reduced pressure to dryness. The residue was dissolved in ethanol,heated to reflux until the reaction was complete. The mixture wasevaporated to dryness. The crude residue was purified by silica gelcolumn chromatography to afford ethylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)glycinate (70 mg) as yellow oil. The product was dissolved inmethanol/H₂O (4 ml/ml), and then lithium hydroxide monohydrate (20 mg)was added. After stirring at room temperature for 2 h, a few drops ofacetic acid were added to the mixture in an ice bath to adjust the pH toacidity. The mixture was filtrated under reduced pressure to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)glycine (45 mg) as a white solid. ¹H NMR (400 MHz, DMSO) δ 8.73 (d,J=1.7 Hz, 1H, —ArH), 8.55 (dd, J=4.8, 1.6 Hz, 1H, DMSO), 7.96 (dt,J=7.9, 1.9 Hz, 1H, DMSO), 7.65 (dd, J=7.6, 1.6 Hz, 1H, —ArH), 7.55-7.35(m, 9H, —ArH), 7.12 (s, 1H, —ArH), 5.33 (s, 2H, —CH₂—), 5.29 (s, 2H,—CH₂—), 3.91 (s, 2H, —CH₂—), 3.11 (s, 2H, —CH₂—). MS (FAB): 569 (M+1).

Example 13N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)valine

The procedure was the same as in Example 12, except that ethyl ester ofvaline was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)valine as a white solid. ¹H NMR (400 MHz, DMSO) δ 8.69 (s, 1H, —ArH),8.56 (d, J=3.9 Hz, 1H, —ArH), 7.88 (d, J=7.8 Hz, 1H, —ArH), 7.66 (d,J=7.4 Hz, 1H, —ArH), 7.55-7.33 (m, 9H, —ArH), 7.06 (s, 1H, —ArH), 5.31(s, 2H, —CH₂—), 5.22 (d, J=11.2 Hz, 2H, —CH₂—), 3.52 (s, 2H, —CH₂—),2.91 (d, J=6.4 Hz, 1H, —CH—), 1.80 (dq, J=13.2, 6.5 Hz, 1H, —CH—), 0.86(dd, J=18.9, 6.7 Hz, 6H, —CH₃). MS (FAB): 611 (M+1).

Example 14(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino) but-2-enenitrile

The procedure was the same as in Example 12, except that(E)-3-aminobut-2-enenitrile was used in place of ethyl ester of glycinewithout hydrolysis to afford(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino) but-2-enenitrile as a white solid. ¹H NMR (400 MHz,) δ 8.70(s, 1H, —ArH), 8.55 (d, J=4.2 Hz, 1H, —ArH), 7.99-7.81 (m, 1H, —ArH),7.65 (d, J=7.0 Hz, 1H, —ArH), 7.58-7.31 (m, 9H, —ArH), 7.11 (d, J=33.8Hz, 1H), 6.30 (s, 1H, ═CH), 5.28 (d, J=14.1 Hz, 4H, —CH₂—), 3.27 (s, 2H,—CH₂—), 2.21-1.96 (m, 3H). MS (FAB): 576 (M+1).

Example 15 N,N-bis(2-hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

The procedure was the same as in Example 12, except thatbis(2-hydroxyethyl)amine was used in place of ethyl ester of glycinewithout hydrolysis to afford N,N-bis(2-hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamineas a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (s, 1H, ArH),8.58-8.51 (m, 1H, ArH), 7.88 (d, J=7.2 Hz, 1H, ArH), 7.65 (d, J=7.6 Hz,1H, ArH), 7.56-7.32 (m, 9H, ArH), 7.05 (d, J=2.4 Hz, 1H, ArH), 5.29 (s,2H, —OCH₂—), 5.24 (s, 2H, —OCH₂—), 4.35 (s, 2H, —OH), 3.58 (s, 2H,—CH₂—), 3.41 (br s, 4H, —CH₂—), 2.51 (m, 4H, —CH₂—, overlapped insolvent peak). MS (FAB): 598 (M+1).

Example 16N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

The procedure was the same as in Example 12, except thatN-(2-aminoethyl)methanesulfonamide was used in place of ethyl ester ofglycine without hydrolysis to affordN-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamineas a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H, ArH), 8.55(d, J=4.4 Hz, 1H, ArH), 7.89 (d, J=7.6 Hz, 1H, ArH), 7.65 (d, J=8.0 Hz,1H, ArH), 7.56-7.33 (m, 9H, ArH), 7.07 (s, 1H, ArH), 6.95 (s, 1H, —NH—),5.30 (s, 2H, —OCH₂—), 5.25 (s, 2H, —OCH₂—), 3.64 (s, 2H, —CH₂—), 3.01(m, 2H, —CH₂—), 2.88 (s, 3H, —CH₃), 2.59 (t, J=6.4 Hz, 2H, —CH₂—). MS(FAB): 631 (M).

Example 17N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

The procedure was the same as in Example 12, except thatN-(2-aminoethyl)acetamide was used in place of ethyl ester of glycinewithout hydrolysis to affordN-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamineas a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H, ArH), 8.55(d, J=4.0 Hz, 1H, ArH), 7.88 (d, J=8.0 Hz, 1H, ArH), 7.80 (t, J=6.0 Hz,1H, —NH—), 7.65 (d, J=6.0 Hz, 1H, ArH), 7.57-7.35 (m, 9H, ArH), 7.07 (s,1H, ArH), 5.30 (s, 2H, —OCH₂—), 5.25 (s, 2H, —OCH₂—), 3.65 (s, 2H,—CH₂—), 3.12 (q, J=6.0 Hz, 2H, —CH₂—), 2.54 (t, J=6.4 Hz, 2H, —CH₂—),1.78 (s, 3H). MS (FAB): 595 (M).

Example 18(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino) but-2-enoic acid

The procedure was the same as in Example 12, except that(E)-3-aminobut-2-enoic acid was used in place of ethyl ester of glycinewithout hydrolysis to afford(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino)but-2-enoic acid as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (s,1H, ArH), 8.55 (d, J=4.4 Hz, 1H, ArH), 7.98 (d, J=8.0 Hz, 1H, ArH), 7.65(dd, J₁=7.6 Hz, J₂=1.2 Hz, 1H, ArH), 7.55-7.34 (m, 9H, ArH), 7.13 (s,1H, ArH), 5.33 (s, 2H, —OCH₂—), 5.29 (s, 2H, —OCH₂—), 3.85 (dd, J₁=52.4Hz, J₂=13.2 Hz, 2H), 3.05 (s, 1H, ═CH), 1.12 (d, J=6.4 Hz, 3H). MS(FAB): 594 (M+1).

Example 192-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino)ethanesulfonic acid

The procedure was the same as in Example 12, except that2-aminoethanesulfonic acid was used in place of ethyl ester of glycinewithout hydrolysis to afford2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino)ethanesulfonic acid as a white solid. ¹H NMR (400 MHz, DMSO)δ 8.79 (s, 1H), 8.62 (d, J=4.8 Hz, 1H), 8.42 (d, J=7.2 Hz, 1H), 7.85 (t,J=4.8 Hz, 1H), 7.43 (m, 9H), 7.04 (s, 1H), 5.37 (s, 2H), 5.24 (s, 2H),4.33 (s, 2H), 3.39 (m, 2H), 2.97 (m, 2H), 2.65 (br, 1H). MS (FAB): 618(M+1).

Example 20N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)leucine

The procedure was the same as in Example 12, except that ethyl ester ofleucine was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)leucine as a white solid. ¹H NMR (400 MHz, DMSO) δ 8.69 (s, 1H), 8.52(d, J=4.8 Hz, 1H), 8.42 (d, J=7.2 Hz, 1H), 7.85 (t, J=4.8 Hz, 1H), 7.43(m, 9H), 7.04 (s, 1H), 5.37 (s, 2H), 5.24 (s, 2H), 3.79 (m, 2H), 3.18(m, 1H), 1.86 (m, 1H), 1.41 (m, 2H), 0.74 (m, 6H). MS (FAB): 624 (M).

Example 21N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)tyrosine

The procedure was the same as in Example 12, except that ethyl ester oftyrosine was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)tyrosine as a white solid. ¹H NMR (400 MHz, DMSO) δ 8.62 (s, 1H), 8.50(d, J=3.2 Hz, 1H), 7.81 (t, J=7.6 Hz, 1H), 7.60 (t, J=9.2 Hz, 1H), 7.38(m, 8H), 7.21 (s, 1H), 7.00 (s, 1H), 6.93 (d, J=8.0 Hz, 2H), 6.59 (d,J=8.0 Hz, 2H), 5.25 (s, 2H), 5.17 (s, 2H), 3.64 (m, 2H), 3.21 (t, J=6.8Hz, 1H), 2.78 (m, 2H). MS (FAB): 674 (M).

Example 22N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)isoleucine

The procedure was the same as in Example 12, except that ethyl ester ofisoleucine was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)isoleucine as a white solid. ¹H NMR (400 MHz, DMSO) δ 8.64 (s, 1H), 8.50(d, J=4.8 Hz, 1H), 8.38 (d, J=7.2 Hz, 1H), 7.82 (t, J=4.8 Hz, 1H), 7.43(m, 9H), 7.00 (s, 1H), 5.25 (s, 2H), 5.18 (s, 2H), 3.63 (m, 2H), 3.48(m, 1H), 2.28 (m, 1H), 1.50 (m, 2H), 1.06 (m, 3H), 0.73 (m, 3H). MS(FAB): 624 (M).

Example 23N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)asparagine

The procedure was the same as in Example 12, except that ethyl ester ofasparagine was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)asparagine as a white solid. ¹H NMR (400 MHz, DMSO) δ11.96 (s, 1H), 8.72(s, 1H), 8.52 (d, J=8.0 Hz, 1H), 8.16 (s, 1H), 7.68 (t, J=8.0 Hz, 1H),7.44 (m, 10H), 7.01 (m, 2H), 5.26 (s, 2H), 5.21 (s, 2H), 3.65 (m, 2H),3.35 (m, 1H), 2.65 (m, 2H). MS (FAB): 625 (M+1).

Example 24N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

The procedure was the same as in Example 12, except that2-aminoethan-1-ol was used in place of ethyl ester of glycine withouthydrolysis to affordN-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamineas a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 1H, Ar—H), 8.54(d, J=15.4 Hz, 1H, Ar—H), 7.92 (s, 1H, Ar—H), 7.72-7.25 (m, 10H), 7.11(s, 1H, Ar—H), 5.30 (s, 2H, —CH₂—), 5.28 (s, 2H, —CH₂—), 3.89 (m, 2H,—CH₂—), 3.54 (s, 2H, —CH₂—), 2.76 (s, 2H, —CH₂—). MS (FAB): 554 (M+1).

Example 25N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)alanine

The procedure was the same as in Example 12, except that ethyl ester ofalanine was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)alanine as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H,Ar—H), 8.53 (m, 1H, Ar—H), 7.93 (d, J=8.0 Hz, 1H, Ar—H), 7.61 (d, J=8.1Hz, 1H, Ar—H), 7.53-7.31 (m, 9H, Ar—H), 7.08 (s, 1H, Ar—H), 5.29 (s, 2H,—CH₂—), 5.27-5.23 (m, 2H, —CH₂—), 3.96-3.77 (m, 2H, —CH₂—), 3.12 (m, 1H,—CH—), 1.19 (d, J=7.0 Hz, 3H, —CH₃). MS (FAB): 582 (M+1).

Example 26N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)proline

The procedure was the same as in Example 12, except that ethyl ester ofproline was used in place of ethyl ester of glycine to affordN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)proline as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (s, 1H, ArH),8.56 (d, J=6.0 Hz, 1H, ArH), 7.95 (d, J=8.0 Hz, 1H, ArH), 7.66 (d, J=7.6Hz, 1H, ArH), 7.5-7.34 (m, 9H, ArH), 7.12 (s, 1H, ArH), 5.35-5.27 (m,4H, —OCH₂—), 3.95 (dd, J₁=51.6 Hz, J₂=13.2 Hz, 2H, —CH₂—), 3.12 (m, 1H,—CH—), 2.67 (m, 1H, —CH₂—), 2.07 (m, 1H, —CH₂—), 1.89 (m, 1H, —CH₂—),1.78 (m, 1H, —CH₂—), 1.68 (m, 1H, —CH₂—), 0.84 (m, 1H, —CH₂—). MS (FAB):608 (M).

Example 27 (S)-SodiumN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine (299 mg) was dissolved in 1 ml of 0.5 N sodium hydroxideaqueous solution. After being stirred for 30 min at room temperature,absolute ethanol was added to the solution until solids appeared. Afterbeing heated to dissolve and cooled to room temperature, the mixture wasplaced in the refrigerator for freezing. Then the mixture was filteredto afford the product (300 mg) as a white solid. ¹H NMR (400 MHz,Methanol-d4) δ 8.65 (s, 1H, —ArH), 8.49 (d, 1H, —ArH), 8.04 (d, 1H,—ArH), 7.63 (d, J=8.0 Hz, 1H, —ArH), 7.50-7.34 (m, 8H, —ArH), 7.31 (d,J=7.5 Hz, 1H, —ArH), 6.84 (s, 1H, —ArH), 5.28 (s, 2H, —CH₂—), 5.23 (s,2H, —CH₂—), 3.85-3.74 (m, 2H, —CH₂—), 3.72 (d, J=4.6 Hz, 1H, —CH₂—),3.67 (m, 1H, —CH₂—), 3.15 (t, J=5.6 Hz, 1H, —CH—). MS (FAB): 599 (M+1).

Example 28 (S)-CalciumN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

(S)-SodiumN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate (243 mg) was dissolved in 5 ml of water. Aqueous solution ofcalcium dichloride (1%, 2.22 ml) was added dropwise while being stirredat room temperature. Then the mixture was stirred overnight, filtered,washed with water, and dried to afford the product (240 mg) as a whitesolid. MS (FAB): 599 (M+1).

Example 29 (S)-(5-methyl-2-oxo-1,3-dioxol-4-yl)methylN-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate

(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine (299 mg) and 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one(74 mg) were dissolved in 10 ml of DMF. After a catalytic amount ofpotassium iodide was added, the mixture was stirred at 30° C. until thereaction was completed. The mixture was poured into a stirred ice-coldsaturated aqueous solution of sodium bicarbonate, then filtered, and thesolid was washed with water and dried to afford the product (98 mg). MS(FAB):711 (M+1).

Pharmacological Experiments

1. In vitro activity evaluation: Cisbio PD-1/PD-L1 binding assay kit wasapplied for the detection method of in vitro enzymology level.

Screening principles and methods of PD-1/PD-L1 small molecule inhibitors

1) Principle: PD-1 protein is with HIS tag, and PD-1 ligand PD-L1 iswith hFc tag. Eu labeled anti-hFc antibody and XL665 labeled anti-HISantibody are combined with the above two label proteins respectively.After laser excitation, energy can be transferred from donor Eu toreceptor XL665, allowing XL665 to glow. After adding inhibitors(compounds or antibodies), blocking the binding of PD-1 and PD-L1 makesthe distance between Eu and XL665 far away, the energy can not betransferred, and XL665 does not glow.2) Experimental method: The specific method can be referred to Cisbio'sPD-1/PD-L1 Kit (item 64CUS000C-2). Reagents should be dispensed in thefollowing order. For 384-well white ELISA plate, 2 μl of diluent ortarget compound diluted with diluent was added to each well, and then 4μl of PD-1 protein and 4 μl of PD-L1 protein were added per well,incubated for 15 min at room temperature; and 10 μl of a mixture ofanti-Tag1-Eu3⁺ and anti-Tag2-XL665 was added per well and incubated for1 h to 4 h at room temperature and the fluorescence signals at 665 nmand 620 nm were measured with an Envison instrument. HTRF rate=(665nm/620 nm)*10⁴. 8-10 concentrations were detected for each compound andIC₅₀ was calculated by Graphpad software.3) The results of the screening were shown in Table 1.

TABLE 1 Evaluation of the inhibitory activity of the example compoundsat molecular level on the interaction between PD-1 and PD-L1: ExampleIC₅₀ (M) 1 1.48 × 10⁻⁷ 4 <10⁻¹³ 6 8.23 × 10⁻⁸ 8 4.29 × 10⁻⁸ 9 4.01 ×10⁻⁸ 10 1.34 × 10⁻⁷ 11 3.18 × 10⁻⁷ 12 4.11 × 10⁻⁸ 13 2.00 × 10⁻⁷ 14 2.69× 10⁻⁵ 15 5.10 × 10⁻⁸ 16 1.99 × 10⁻⁷ 17 5.51 × 10⁻⁷ 18 2.33 × 10⁻⁹ 191.62 × 10⁻⁵ 20 6.10 × 10⁻⁸ 21 4.06 × 10⁻⁷ 22 4.99 × 10⁻⁶ 23 8.35 × 10⁻⁷24 5.38 × 10⁻⁸ 25 5.29 × 10⁻⁹ 26 10⁻¹²~10⁻¹³

Cisbio HTRF detection showed that the interaction of PD-1 and PD-L1could be significantly inhibited by the Example 4 compound at themolecular level, with IC₅₀<10⁻¹³ mol/L.

2. The Example 4 compound's capacity of relieving the inhibition of IFNγby ligand PD-L1:

The expression level of IFNγ can reflect the proliferative activity of Tlymphocytes. Using the extracted human PBMC (peripheral bloodmononuclear cell), on the basis that T lymphocyte could be activated bythe anti-CD3/anti-CD28 antibody, the ligand PD-L1 was added to theinhibit T lymphocyte, the example compounds' capacity of relieving theinhibition by the PD-L1 was investigated.

The specific procedure is as follows. DAKEWE human lymphocyte separationsolution (DKW-KLSH-0100) was used to extract PBMC from human wholeblood, and PBMC was inoculated into 96 well plate, with 3×10⁵ cells perwell. Human PD-L1 protein (final concentration 5 μg/ml),anti-CD3/anti-CD28 antibody (final concentration 1 μg/ml) andproportional dilution of the Example 4 compound were added respectively.After 72 h, the expression level of IFNγ in the supernatant was detectedby Cisbio IFNγ test kit. The experimental results showed that theinhibition of PD-L1 to expression level of IFNγ could be partiallyrelieved by the Example 4 compound (YPD29B) at 10 nM, and the level ofIC₅₀ was determined as the level of 1.8×10⁻¹⁰ mol/L by testing differentconcentrations (FIG. 1).

3. The efficacy of the Example 4 compound in vivo

The methods of pharmacodynamics were as follows:

The method in subcutaneous xenograft tumor was as follows. The culturedspecific tumor cells were digested and collected by centrifugation, andwashed with sterile physiological saline for two times and then counted.The cell concentration was adjusted to 5×10⁶/ml by physiological saline,and 0.2 ml of cell suspension was inoculated to the right armpit ofC57BL/6 or Bablc mice. After inoculation, the animals were randomlydivided into two groups in next day. Each group had 6-7 mice. Afterweighing, the animals were dosed once each day to monitor tumor size.When the tumor size reached to a certain size, the mice was weighed andblood was collected from mice orbit and then the mice were killed byremoving the neck. The tumor tissue, thymus tissue and spleen tissuewere collected and weighed respectively. Finally, the tumor growthinhibition rate was calculated, and the tumor growth inhibition rate wasused to evaluate the level of anti-tumor effect.

The method in B16F10 lung metastasis model was as follows. The culturedB16F10 tumor cells were digested and centrifuged and washed for twotimes with sterile physiological saline and then counted. And the cellconcentration was adjusted to 2.5×10⁶/ml by physiological saline. 0.2 mlof cells were injected into the C57BL/6 mice through the tail vein, andthe tumor cells will gather in the lung of the mice. After inoculation,the animals were randomly divided into two groups in next day. Eachgroup had 6-7 mice. After weighing, the animals were dosed once eachday. After 3 weeks, the mice were weighed and killed, the lung tissuewas collected and weighed, and the number of lung tumors was countedafter being fixed by the Bouin's Fluid. Finally, the tumor growthinhibition rate was calculated, and the tumor growth inhibition rate wasused to evaluate the level of anti-tumor effect.

The method in Lewis lung cancer hydrothorax model was as follows: Thesubcutaneous xenograft tumor of Lewis lung cancer was homogenized andwashed for two times with sterile physiological saline, and the cellconcentration was adjusted to 2.5×10⁵/ml by physiological saline. 0.2 mlof cells were injected into the thoracic cavity of C57BL/6 mice. Afterinoculation, the animals were randomly divided into two groups in nextday. Each group had 6-7 mice. After weighing, the animals were dosedonce each day. Animals were sacrificed when the weight of the animals inthe control group suddenly dropped. The liquid in thoracic cavity wasextracted with syringe and the volume of fluid was recorded.

In the study of the mechanism of the above models, the method of flowcytometry was adopted in measuring the total cell proportion of T cellsof various types. The specific steps were as follows. The samples weretreated at first. For blood tissue, the orbital blood was taken. The redcell lysate was used to remove the red blood cells, and then the PBSbuffer was used for wash. After being washed, the cells were collected.For the tumor and spleen, the tissues were grinded with a homogenizer,and then diluted with PBS buffer, then filtered by 300 meshes of screen.After the number of cells was counted for each sample, 1×10⁶ cells wereadded into EP tube and stained for flow antibody. After incubation for 1h on ice, each sample was washed 2 times with PBS buffer. The cellpopulation was analyzed by VERSE flow instrument of BD Company. Thetotal number of cells in tumor tissue was 1×10⁵ and the total number ofcells in blood and spleen tissues was 1×10⁴. The ratio of T cells tototal number of cells was analyzed after flow cytometry.

(1) Subcutaneous Xenograft Model of High Metastatic Melanoma B16F10

For the high metastatic melanoma B16F10, the example compounds (45 mg/kgof Example 5 compound, 15 mg/kg of hydrochloride form of Example 4compound and 15 mg/kg of sodium salt of Example 4 compound) cansignificantly inhibit the growth of the subcutaneous tumor, with therespect of tumor volume or weight (FIG. 2, FIG. 3 and Table 2) and therate of inhibition of tumor weight can be 45.27%, 38.37% and 64.11%respectively.

TABLE 2 Inhibition of Bl6F10 subcutaneous xenograft tumors by Examplecompounds Number Body weight (g) (Begin/ Mean ± SD Tumor weight(g) GroupDose End) Begin End X ± SD T/C % (TGI %) Vehicle Control 6/6 20.3 ± 1.023.7 ± 1.7 2.58 ± 1.56 NA Cyclophos- 80 mg/kg 5/5 20.3 ± 0.8 23.3 ± 1.41.55 ± 0.59  60.00(40.00) phamide (CTX) Example 5 15 mg/kg 5/5 20.4 ±1.0 24.2 ± 2.2 2.69 ± 1.99 104.26(−4.26) 45 mg/kg 5/5 20.3 ± 1.0 22.6 ±1.4 1.41 ± 0.60  54.73(45.27) Example 4 15 mg/kg 5/5 20.3 ± 0.4 21.8 ±2.1 0.93 ± 0.89  35.89(64.11)* sodium salt 45 mg/kg 5/5 20.4 ± 0.9 23.8± 1.4 2.57 ± 0.85  99.77(0.23) Example 4 15 mg/kg 5/5 20.3 ± 0.6 22.6 ±1.4 1.59 ± 0.91  61.63(38.37) hydrochloride 45 mg/kg 5/5 20.3 ± 0.7 23.1± 1.7 2.19 ± 0.92  84.81(15.19) form T/C: Relative tumor proliferationrate TGI: Tumor growth inhibition rate NA: Not applicable *P < 0.05 vsVehicle control

From the analysis of mechanism, it can be seen that Example 5 compound,sodium salt of Example 4 compound and hydrochloride form of Example 4compound can increase the proportion of tumor-infiltrating lymphocytes(FIG. 4, Table 3) and sodium salt of Example 4 compound can increase theproportion of lymphocytes in the spleen samples (FIG. 5, Table 4).

TABLE 3 Effects of Example 4 and Example 5 on tumor-infiltrating Tlymphocytes Group CD3+ (%) CD4+ (%) CD8+ (%) Vehicle control  6.5 ± 0.84.8 ± 3.7 3.4 ± 0.1 Cyclophosphamide (CTX)  3.6 ± 1.5 1.7 ± 0.4 1.4 ±0.3 Example 5 45 mg 10.1 ± 4.5 9.0 ± 4.7 5.2 ± 2.8 Example 4 sodium salt15 mg 13.3 ± 6.9 7.2 ± 3.4 3.9 ± 1.4 Example 4 hydrochloride form 15.2 ±3.9 15.0 ± 10.7 9.6 ± 3.6 15 mg

TABLE 4 Effects of sodium salt of Example 4 compound on T lymphocytes inspleen Group CD3+ (%) CD4+ (%) CD8+ (%) Vehicle control 62.5 ± 7.6 21.3± 4.0  9.6 ± 2.1 Cyclophosphamide (CTX) 78.6 ± 2.5 24.6 ± 2.6 15.2 ± 3.1Example 4 sodium salt 15 mg 74.3.3 ± 3.5   27.0 ± 1.8 13.6 ± 1.8

(2) Lung Metastasis Model of High Metastatic Melanoma B16F10

For metastatic lung cancer models with high metastatic melanoma B16F10,the sodium salt of Example 4 compound can significantly inhibit thenumber of lung metastases at 15 mg/kg dose (FIG. 6, Table 5).

TABLE 5 Example compounds' inhibition effect on Pulmonary metastasismodel of Bl6F10 Body weight(g) Tumor number Number Mean ± SD T/C % GroupDose (End/Begin) Begin End X ± SD (TGI %) Vehicle control 6/6 20.2 ± 0.421.0 ± 0.3 21 ± 15 NA Cyclophosphamide 80 mg/kg 5/5 20.1 ± 0.8 21.4 ±0.5 18 ± 14 85.7(14.3) (CTX) Example 5 15 mg/kg 5/5 20.6 ± 0.7 20.2 ±1.5 18 ± 18 85.7(14.3) 45 mg/kg 5/5 20.5 ± 0.6 21.2 ± 0.7 16 ± 7 76.2(23.8) Example 4 sodium salt 15 mg/kg 5/5 20.1 ± 0.9 21.5 ± 1.2 12 ±3  57.1(42.3) 45 mg/kg 5/5 20.3 ± 0.6 21.4 ± 0.3 19 ± 15 90.5(9.5)Example 4 15 mg/kg 5/5 20.5 ± 0.6 21.3 ± 1.0 14 ± 10 66.7(33.3)hydrochloride form 45 mg/kg 5/5 20.6 ± 1.0 21.3 ± 0.4 17 ± 12 81.0(19.0)T/C: Relative tumor proliferation rate TGI: Tumor growth inhibition rateNA: Not applicable *P < 0.05 vs Vehicle control

From analysis of the mechanism, it can be seen Example 4 and Example 5could increase the percentage of lymphocyte in mouse blood (FIG. 7,Table 6).

TABLE 6 Example compounds' effect on the percentage of T ymphocyte inmouse blood Group CD3+ CD4+ CD8+ Vehicle control 21.0 ± 2.6 12.3 ± 2.17.0 ± 1.1 Cyclophosphamide (CTX) 22.4 ± 5.5 13.0 ± 2.4 7.5 ± 2.4 Example5 15 mg 22.7 ± 4.8 14.4 ± 3.3 7.4 ± 1.9 Example 5 45 mg 25.8 ± 3.0 15.7± 2.5 7.6 ± 1.8 Example 4 sodium salt 15 mg 29.0 ± 3.7 17.8 ± 2.4 9.6 ±0.8 Example 4 sodium salt 45 mg 23.2 ± 3.6 14.7 ± 2.5 7.6 ± 1.3 Example4 hydrochloride 15 mg 29.3 ± 2.9 18.6 ± 1.6 10.7 ± 1.3  Example 4hydrochloride 45 mg 26.8 ± 4.1 17.4 ± 2.0 8.5 ± 2.3

(3) Subcutaneous Xenograft Model of Mouse Breast Cancer EMT6

For subcutaneous xenograft model of mouse breast cancer EMT6, sodiumsalt of Example 4 compound has some inhibition effect on mouse breastcancer EMT6. At the dose of 10 mg and 15 mg, sodium salt of Example 4compound has 20% and 22% inhibition effect respectively (FIG. 8, Table6). In addition, the combination of sodium salt of Example 4 compoundand Cyclophosphamide can significantly increase the tumor growthinhibition rate of Cyclophosphamide from 85% to 95% (FIG. 8, Table 7).

TABLE 7 Example 4 compound's inhibition effect on mouse subcutaneousxenograft of EMT6 Number Body weight (g) (End/ Mean ± SD Tumorweight(g)Group Dose Begin) Begin End X ± SD T/C % (TGI %) Vehicle 6/6 18.4 ± 0.320.3 ± 0.3 1.72 ± 0.22  NA control Cyclophosphamide  60 mg/kg 5/5 18.4 ±0.3 19.4 ± 0.7* 0.25 ± 0.17*** 14.1(85.9)*** Cyclophosphamide +  60mg/kg + 5/5 17.8 ± 1.1 18.9 ± 0.4*** 0.08 ± 0.04***  4.8(95.2)***Example 4  10 mg/kg sodium salt Example 4 1.5 mg/kg 6/6 18.4 ± 0.9 22.0± 1.6  1.5 ± 0.61 86.9(13.1) sodium salt   5 mg/kg 6/6 18.4 ± 0.4 21.0 ±1.6 1.37 ± 0.23* 79.8(20.2)*  10 mg/kg 6/6 18.4 ± 0.6 21.6 ± 1.3 1.34 ±0.34* 77.7(22.3)*  15 mg/kg 6/6 18.5 ± 0.4 22.0 ± 1.1* 1.47 ± 0.6585.3(14.7) T/C: Relative tumor proliferation rate TGI: Tumor growthinhibition rate NA: Not applicable *P < 0.05 vs Vehicle control

(4) Mouse Lewis Lung Cancer Hydrothorax Model

Sodium salt of Example 4 compound has inhibition effect on mouse Lewislung cancer hydrothorax model. The hydrothorax incidence rate in vehiclecontrol group was 75%, whereas at the dose of 10 mg, sodium salt ofExample 4 compound can reduce the rate to 33% (Table 8). The mean volumeof the hydrothorax of mice was 0.3 ml in the vehicle control group andin the group administrated with sodium salt of Example 4 compound themouse only had 0.2 ml of hydrothorax (FIG. 9, Table 9). Further, sodiumsalt of Example 4 compound can significantly increase thymus index (FIG.10).

TABLE 8 Example 4's effect on hydrothorax incidence rate of Lewis lungcancer Vehicle control Example 4 sodium salt 10 mg 75% 33%

TABLE 9 Example 4's effect on hydrothorax volume of Lewis lung cancerVehicle control Example 4 sodium salt 10 mg 0.3 ml 0.2 ml

(5) Subcutaneous Xenograft Model of Mouse Colon Cancer MC38

For subcutaneous xenograft model of mouse colon cancer MC38, sodium saltof Example 4 compound has significant inhibition effect. Furthermore,sodium salt of Example 4 compound has a synergistic antitumor effect onthis cancer with Cyclophosphamide (CTX) (FIG. 11, Table 10).

TABLE 10 Example compounds' effect on subcutaneous xenograft model ofmouse colon cancer MC38 Body weight (g) Number Mean ± SD Tumor weight(g)Group Dose (End/begin) Begin End Mean ± SD T/C % (TGI %) Vehicle 6/617.9 ± 0.5 22.3 ± 1.1 3.18 ± 0.82 NA control Cyclophosphamide  60 mg/kg6/6 17.9 ± 0.6 19.1 ± 0.8*** 0.17 ± 0.05***  5.4(94.6)***Cyclophosphamide +  60 mg/kg + 6/6 18.2 ± 0.6 18.8 ± 0.8*** 0.06 ±0.04***  1.9(98.1)*** Example 4  10 mg ## ## sodium salt Example 4 2.5mg/kg 6/6 18.0 ± 0.4 21.9 ± 0.7 3.25 ± 0.61 −2.3(102.3)   5 mg/kg 6/618.2 ± 0.3 22.8 ± 0.6 2.78 ± 0.44 87.3(12.7)  10 mg/kg 6/6 18.2 ± 0.321.5 ± 0.8 2.14 ± 0.78* 67.3(32.7)*  20 mg/kg 6/6 18.2 ± 0.4 20.9 ± 1.11.87 ± 0.90* 58.8(41.2)* T/C: Relative tumor proliferation rate TGI:Tumor growth inhibition rate NA: Not applicable *P < 0.05 vs Vehiclecontrol; ***P < 0.001 vs Vehicle control; ##p < 0.01 vs Cyclophosphamide(CTX)4. The interaction of Example 4 compound/PD-L1 antibody with PD-L1protein was tested by Biacore

(1) Experimental Principle

Surface plasmon is a kind of electromagnetic wave on the surface ofmetal, produced by the interaction of photon and electron in freevibration. Surface plasmon resonance (SPR) is an optical phenomenon thatoccurs on the surface of two kinds of media, which can be induced byphoton or electron. The phenomenon of total reflection of light fromlight dense medium into light scattering medium will form evanescentwave into light scattering medium. When the total reflected evanescentwave meets the plasma wave on the metal surface, the resonance mayoccur, and the energy of reflected light decreases and the resonancepeak appears on the reflected light energy spectrum. This resonance iscalled the surface plasmon resonance. The incident angle of the surfaceplasmon resonance is called the SPR angle. The SPR biosensor provides asensitive, real-time, non-label detection technique for monitoring theinteraction of molecules. The sensor detects the change of the SPRangle, and SPR is also related to the refractive index of the metalsurface. When an analyte is bond on the surface of the chip, it leads tothe change of the refractive index of the chip surface, which leads tothe change of the SPR angle. This is the basic principle of thereal-time detection of intermolecular interaction by the SPR biosensor.In the interaction analysis, the change of SPR angle is recorded on thesensor map in real time.

(2) Experimental Methods

The PD-L1 protein was captured on the Fc4 channel of NTA chip by capturemethod, and the buffer system was PBS-P+, pH7.4, 0.01% DMSO. A series ofconcentration of compounds and PD-L1 antibodies were prepared and flowedthrough the surface of the chip for the determination of interaction.

(3) Experimental Results

TABLE 11 The affinity of Example 4 compound and PD-L1 antibody to PD-L1Ligand Analyte ka (1/Ms) kd (1/s) KD (M) PD-L1 protein PD-L1 antibody2.016E+5 1.358E−4 6.736E−10 PD-L1protein Example 4 1.390E+6 2.815E−52.025E−11

It was preliminarily determined that the binding protein of the Example4 is PD-L1 (FIG. 12). Further Biacore experiments confirmed that thecombination of Example 4 has a strong ability of binding PD-L1 and theaffinity kD value is 2.025E-11 which is even stronger than that of theantibody of PD-L1 (Table 11, FIG. 12-14).

1. A nicotinyl alcohol ether derivative of Formula (I):

or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R₁ is selected from

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄ alkyl, ethenyl, trifluoromethyl, methoxy.
 2. A nicotinyl alcohol ether derivative of claim 1, represented by formula (IA), or a pharmaceutically acceptable salt or a stereoisomer thereof;

wherein: R₁ is selected from

R₃ is selected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄ alkyl, ethenyl, trifluoromethyl, and methoxy.
 3. A nicotinyl alcohol ether derivative of claim 2, represented by formula (IA-1), or a pharmaceutically acceptable salt or a stereoisomer thereof;

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄ alkyl, ethenyl, trifluoromethyl, and methoxy.
 4. A nicotinyl alcohol ether derivative of claim 2, represented by formula (IA-2), or a pharmaceutically acceptable salt, or a stereoisomer thereof:

wherein: R₃ is selected from substituted C₁-C₈ saturated alkylamino, substituted C₂-C₆ unsaturated alkylamino, substituted N-containing C₂-C₆ heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, amino, C₁-C₆ alkylamino, acetylamino, cyano, ureido (—NH(C═O)NH₂), guanidino (—NH(C═NH)NH₂), ureido amino (—NH—NH(C═O)NH₂), guanidino amino (—NH—NH(C═NH)NH₂), sulfonylamino (—NHSO₃H), sulfamoyl (—SO₂NH₂), methanesulfonylamino (—NH—SO₂CH₃), hydroxyformyl (—COOH), C₁-C₈ alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl,

X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C₁-C₄ alkyl, ethenyl, trifluoromethyl, and methoxy.
 5. A nicotinyl alcohol ether derivative of claim 1, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R₃ is selected from:

wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl; X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.
 6. A nicotinyl alcohol ether derivative of claim 1, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein the compound is selected from: Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

(S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl)serinate

(S)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine

(S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

(S)-isopropyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

(R)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

(R)—N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) valine

(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) hut-2-enenitrile

N, N-bis(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) but-2-enoic acid

2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) ethanesulfonic acid

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) leucine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) tyrosine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) isoleucine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) asparagine

N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) alanine

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) proline

(S)-Sodium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

(S)-Calcium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

(S)-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serinate


7. A nicotinyl alcohol ether derivative of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt comprises a salt formed with an inorganic acid, a salt formed with an organic acid salt, alkali metal ion salt, alkaline earth metal ion salt or a salt formed with organic base which provides a physiologically acceptable cation, and an ammonium salt.
 8. A nicotinyl alcohol ether derivative of claim 7, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, potassium ion; the alkaline earth metal ion is selected from calcium ion and magnesium ion; and the organic base which provides a physiologically acceptable cations is selected form methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl) amine.
 9. A process for the preparation of a nicotinyl alcohol ether derivative of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

for the preparation of the compounds of the formula (I), according to its structure, the preparation method is divided into two steps: (a) 2-hydroxy-4-(2-bromo-3-R1 benzyloxy)-X-substituted benzaldehyde 1 as a starting material is reacted with pyridin-3-yl-methylene halide under basic conditions to obtain an aldehyde-containing intermediate compound 2; (b) the aldehyde-containing intermediate compound 2 as the starting material is condensed with an amino group- or an imino group-containing HR₃ and the resultant product is reduced to obtain a target compound I; wherein R₁, R₃ and X each is defined as claim
 1. 10. A pharmaceutical composition, characterized in that it comprises a nicotinyl alcohol ether derivative of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.
 11. A method for preventing and/or treating a disease associated with the PD-1/PD-L1 signaling pathway in a subject in need of such treatment comprising administering to the subject an effective amount of a nicotinyl alcohol ether derivative of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
 12. The method of claim 11, wherein the disease associated with the PD-1/PD-L1 signaling pathway is selected from cancer, infectious disease, and autoimmune disease.
 13. The method of claim 12, wherein the cancer is selected from skin cancer, lung cancer, urinary tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection and viral infection; the autoimminue disease is selected from the organ specific autoimmune disease and the system autoimmune disease; wherein the organ specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes, severe myasthenia, ulcerative colitis, malignant anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis; the system autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia.
 14. A pharmaceutical composition, characterized in that it comprises a nicotinyl alcohol ether derivative of claim 6, or a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.
 15. A method for preventing and/or treating a disease associated with the PD-1/PD-L1 signaling pathway in a subject in need of such treatment comprising administering to the subject an effective amount of the nicotinyl alcohol ether derivative of claim 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
 16. The method of claim 15, wherein the disease associated with the PD-1/PD-L1 signaling pathway is selected from cancer, infectious disease, and autoimmune disease.
 17. The method of claim 16, wherein the cancer is selected from skin cancer, lung cancer, urinary tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection and viral infection; the autoimminue disease is selected from the organ specific autoimmune disease and the system autoimmune disease; wherein the organ specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes, severe myasthenia, ulcerative colitis, malignant anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis; the system autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia. 